Anticoagulation reversal agent, method for producing the same, and its application

Abstract

The present invention provides an anticoagulant reversal agent which is a compound having the structure represented by formula I, a salt thereof, or a deuterated thereof. Experimental results have shown that the compound according to the present invention can more effectively suppress hemorrhagic complications caused by anticoagulants such as warfarin, heparin, and enoxaparin sodium, and exhibits high efficacy over a broad spectrum.

Description

Cross-reference

[0001] This application claims priority based on a Chinese patent application filed with the China National Intellectual Property Administration on March 24, 2023, with an application number of 202310305256.2 and an invention title of "Anticoagulation Reversal Agent and Its Manufacturing Method, and Its Application", and the entire content thereof is incorporated herein by reference.

Technical Field

[0002] The present invention relates to the technical field of pharmaceutical chemistry, and particularly to an anticoagulation reversal agent and its manufacturing method, and its application.

Background Art

[0003] Currently, approximately 423 million people worldwide suffer from cardiovascular diseases, and a significant number of these patients need to be treated with anticoagulants. Commonly used anticoagulants include vitamin K antagonists, indirect thrombin inhibitors, and direct thrombin inhibitors. 1. Vitamin K antagonists exert an anticoagulant effect by antagonizing vitamin K and reducing the synthesis of prothrombin, factor VIIIX, and factor X in the liver. A typical drug is warfarin. 2. Indirect thrombin inhibitors exert an anticoagulant effect by indirectly inhibiting the activities of factor Xa and factor IIa through interaction with antithrombin. Typical drugs are heparin and low molecular weight heparin. 3. Direct thrombin inhibitors block the final stage of the blood coagulation cascade and thrombus formation by inhibiting thrombin and preventing the degradation of fibrinogen to fibrin. Among them, monovalent thrombin inhibitors (dabigatran etexilate, argatroban) directly inhibit thrombin, while bivalent thrombin inhibitors (bivalirudin, recombinant hirudin) can not only directly inhibit thrombin but also exert an anticoagulant effect by dissociating thrombin from fibrin.

[0004] However, anticoagulation therapy is a double-edged sword; while it can significantly reduce the incidence of thrombotic events as a basic therapy for preventing and treating thrombotic diseases, it can also cause hemorrhagic complications, which in severe cases can be life-threatening. Therefore, there is a clinical need for antagonists that can rapidly and effectively reverse the anticoagulant effect to ensure the safety of patients receiving anticoagulation therapy. Currently, there are few anticoagulant reversals on the market or under development. Examples of reversals available on the market include vitamin K as a reversal agent for warfarin, protamine as a reversal agent for heparin, idarucizumab as a monoclonal antibody against dabigatran etexilate, and Andexxa as an anticoagulant activity reversal agent for the factor Xa inhibitor rivaroxaban / apixaban. Furthermore, patent WO2013082210A1 discloses an anticoagulant reversal agent called diaarginine piperazine (DAP, PER977) that can reverse the anticoagulant effects of heparin, heparin fragments, fondaparinux, and factor Xa inhibitors or factor IIa inhibitors (such as oral factor Xa inhibitors or oral factor IIa inhibitors). DAP is currently undergoing Phase III clinical trials.

[0005] Incidentally, there is currently a clinical need for anticoagulant reversals that exhibit high efficacy across a broad spectrum. [Overview of the Initiative]

[0006] In view of the above circumstances, the present invention aims to solve the technical problem of providing an anticoagulant reversal agent having high efficacy over a wide spectrum, a method for producing the same, and applications thereof.

[0007] The present invention provides a compound having a structure represented by the following formula I, a salt thereof, or a deuterated compound thereof: [ka] In the formula, X, X', and X'' are independently selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups. Y, Y', and Y'' are independently selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups. Z, Z', and Z'' are independently selected from the group consisting of molecular fragments containing heteroatoms that can be protonated under physiological conditions.

[0008] The number of carbon atoms in the alkyl group is preferably 1 to 10, more preferably 1 to 6, and even more preferably 2, 3, 4, or 5.

[0009] The number of carbon atoms in the alkenyl group is preferably 2 to 10, more preferably 2 to 6, and even more preferably 2, 3, 4, or 5.

[0010] The heterocyclic group preferably has 2 to 12 carbon atoms, more preferably a monocyclic heterocyclic group, and even more preferably a 5-membered ring or a 6-membered ring heterocyclic group. The heteroatoms in the heterocyclic group are preferably one or more of N, O, and S.

[0011] The substituents of the alkyl group, alkenyl group, or heteroaryl group are preferably one or more selected independently from the group consisting of amino groups, nitro groups, and halogens, and more preferably amino groups.

[0012] Preferably, YZ, Y'-Z', and Y''-Z'' are independently selected from basic amino acid residues. More preferably, the basic amino acid residues are histidine, arginine, or lysine residues.

[0013] In some specific examples, the aforementioned residue refers to the group remaining after an amino acid has lost its carboxyl group.

[0014] Preferably, the molecular fragment containing the heteroatom is selected from the group consisting of an amino group, a guanidine group, and an imidazole group.

[0015] Preferably, the compound has a structure represented by the following formula I-a.

Chemical formula

[0016] Preferably, the compound has the following structure.

Chemical formula

[0017] Preferably, the compound has the following structure.

Chemical formula

[0018] According to the present invention, a method for producing the above compound including the following steps S1 to S4 is provided: Step S1 of reacting xanthine with a halide represented by the following formula a to obtain Intermediate 1; Step S2 of deprotecting the amino group of Intermediate 1; Step S3 of reacting the deprotected compound with a compound represented by the following formula b to obtain Compound 2; Step S4 of deprotecting Compound 2 to obtain a compound represented by Formula I.

Chemical formula

[0019] According to the present invention, an anticoagulation reversal composition containing the above compound and a pharmaceutically acceptable adjuvant is provided.

[0020] In the present invention, the type of the adjuvant is not particularly limited, and any suitable adjuvant known to those skilled in the art may be used.

[0021] According to the present invention, there is provided the use of the above compound or the above anticoagulation reversal composition in the manufacture of a medicament for preventing, treating or reducing bleeding caused by an anticoagulant.

[0022] Preferably, the anticoagulant is one or more of a vitamin K antagonist and an indirect thrombin inhibitor.

[0023] More preferably, the vitamin K antagonist is warfarin, and the indirect thrombin inhibitor is heparin or low molecular weight heparin. The above anticoagulation reversal agent can also be used in combination with other anticoagulation reversal agents.

[0024] According to the present invention, there is further provided a kit containing the above compound and one or more anticoagulants.

[0025] Preferably, the anticoagulant is one or more of a vitamin K antagonist or an indirect thrombin inhibitor.

[0026] More preferably, the vitamin K antagonist is warfarin, and the indirect thrombin inhibitor is heparin or low molecular weight heparin.

[0027] Compared to the prior art, the present invention provides a compound having the structure represented by formula I, or a salt thereof, or a deuterated compound thereof. Experimental results have shown that the compounds according to the present invention can more effectively suppress bleeding complications caused by anticoagulants such as warfarin, heparin, and enoxaparin sodium, and exhibit high efficacy over a broad spectrum. [Brief explanation of the drawing]

[0028] [Figure 1] This is the mass spectrum of compound 1. [Figure 2] This is the 1H NMR spectrum of compound 1. [Modes for carrying out the invention]

[0029] To further illustrate the present invention, the anticoagulant reversal agent, its manufacturing method, and applications according to the present invention will be described in detail below with reference to examples. [Examples]

[0030] Step 1: [ka]

[0031] Operating instructions: 1. Add 500 mL of DMF to a 1000 mL three-necked flask and start stirring. 2. While controlling the temperature between 0 and 10°C, 27.6g of NaH was slowly added. 3. While controlling the temperature between 0 and 10°C, 21g of xanthine was slowly added. 4. The mixture was heated to room temperature and stirred for 1 hour. 5. Slowly add 115g of N-Boc-bromopropylamine. 6. Stirred overnight at room temperature.

[0032] Post-processing: 7. The reaction mixture was added to 1000 mL of ice water and extracted twice with 1000 mL of dichloromethane (1000 mL x 2). 8. The organic phase was washed twice with 500 mL of saturated saline solution (500 mL x 2). 9. The organic phase was concentrated and dried. 10. The concentrated residue was purified by silica gel column chromatography (eluent: ethyl acetate / methyl tert-butyl ether = 1 / 1). 11. The fraction containing the product was concentrated and allowed to dry. 12. A colorless oily substance was obtained.

[0033] Step 2: [ka]

[0034] Operating instructions: 1. Dioxane was added to a flask containing CG659A oil and dissolved while stirring. 2. The dioxane hydrochloride solution was slowly added dropwise while controlling the temperature to 10±5℃. 3. The mixture was stirred for 16 hours while maintaining a temperature of 20±5℃. 4. The solid product was precipitated.

[0035] Post-processing: 5. The sample was filtered by suction under nitrogen gas protection, and the filter cake was washed with dioxane (25 mL x 2). 6. The filtered cake was added to methyl tert-butyl ether (150 mL) and stirred at room temperature for 1 hour. 7. The sample was filtered by suction under nitrogen gas protection, and the filter cake was washed with methyl tert-butyl ether (25 mL).

[0036] Step 3: [ka]

[0037] Operating instructions: Boc-L-Arg(Pbf)-OH (CG659C, 2.0g, 3.3eq) and DMF (30mL) were added to a 1100mL three-necked flask, and DIPEA (1.5g, 10eq) was added while stirring. The mixture was cooled to 2.0±5℃, HATU (1.5g, 3.3eq) was added, and the mixture was kept warm for 30 minutes. 3. The temperature was controlled to 0±5℃, and CG659-B (0.5g, 1.0eq) was added. 4. The mixture was heated to room temperature and allowed to react overnight (16 hours). 5. Water (30 mL) was added to the reaction solution to precipitate the solid, which was then collected by suction and filtration. 6. Ethyl acetate was added to dissolve the product, washed with water, and concentrated to dryness to obtain 1.9 g of a viscous product. 7. The filtrate was extracted with methyl tert-butyl ether (30 mL x 2). 8. Combine the organic phases and wash with water (25 mL). 9. The organic phase was concentrated and dried to obtain 0.3 g of oily substance.

[0038] Step 4: [ka]

[0039] Operating instructions: CG659D / DCM solution (0.5g / 3mL) was added to a 1100mL three-necked flask. 2. TFA (1 mL, 50 eq), water (29.2 mg, 6.0 eq), and TIS (141 mg, 3.3 eq) were added at room temperature. 3. Stir the mixture at room temperature overnight, then add 2 mL of TFA and continue stirring for 5 hours.

[0040] Post-processing: 4. The mixture was concentrated to remove most of the DCM and TFA from the system, yielding 0.7 g of crude product. 5. Add anhydrous ethanol (10 mL) while stirring to precipitate the solid, and stir for 1 hour. 6. The solution was filtered by suction, and the filter cake was rinsed with MTBE (2 mL x 2) to obtain 0.3 g of an off-white solid product. Next, 3 mL of water was added, the pH was adjusted to 8-9 with a 5% NaOH aqueous solution, and the solution was extracted with DCM (10 mL x 2) to obtain 0.06 g of an oily product. Next, the water was removed in several stages with 100 mL of toluene, 50 mL of isopropanol was added, and the solution was dissolved and filtered. The filtrate was spun-dried by centrifugal force to obtain approximately 0.2 g of an oily liquid. This product is denoted as Compound 1.

[0041] Characterization of the structure: The structure of the product was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The results are shown in Figures 1 and 2.

[0042] Mass spectrometry results: [M+H] + The value was 792.5.

[0043] Based on the molecular ion peaks obtained by low-resolution mass spectrometry in Figure 1, the molecular weight of the free base of this product was determined to be 791.5. Furthermore, according to the nitrogen rule, the exact molecular weight of this product is odd, which matches the chemical formula of the target compound. The structure of the product was confirmed by nuclear magnetic resonance (NMRI) results. [Examples]

[0044] Antagonistic effect against warfarin Experimental procedure: Tail amputation bleeding model: SD rats, excluding the control group and the 20 mg / kg + NS control group of compound 1, were force-administered warfarin once daily for 3 consecutive days. 10% chloral hydrate was intraperitoneally injected 30 minutes after the final dose. Anesthetized animals were placed on an electric blanket preheated to 37°C. Within 30 minutes, the jugular vein was isolated, a cannula was inserted, and saline and the drug were injected into the jugular vein. Five minutes after drug administration, an incision was made 5 mm from the tail end of the rat, and the bleeding loss (BL) over 15 minutes was recorded. At the end of the experiment, blood was collected intraperitoneally and the WBCT was measured. Temperature, including room temperature and water bath temperature, was strictly controlled during the experiment.

[0045] Liver laceration bleeding model: SD rats, excluding the control group and the 20 mg / kg + NS control group of compound 1, were force-administered warfarin once daily for 3 consecutive days. 10% chloral hydrate was intraperitoneally injected 30 minutes after the final dose. Anesthetized animals were placed on an electric blanket preheated to 37°C. The jugular vein was isolated within 30 minutes, a cannula was inserted, and the test substance was injected into the jugular vein 30 minutes after aspirin administration. Five minutes after drug administration, the abdominal cavity was opened, and three standard incisions, approximately 1 cm in diameter and 2 mm deep, were made on the liver. Blood seeping from the liver was wiped away with pre-weighed dry cotton, and the bleeding loss (BL) over 30 minutes was recorded.

[0046] The experimental results are shown in Tables 1-3 below. [Table 1]

[0047] [Table 2]

[0048] [Table 3]

[0049] The experimental results revealed the following: Compared to the control group, the solvent group showed a significant increase in induced bleeding in cases of transverse tail amputation (P<0.05), a significant increase in induced bleeding in cases of liver laceration, and a significant prolongation of WBCT (P<0.001). Compared to the solvent group, compound 1 at 20 mg / kg significantly reduced induced bleeding in cases of transverse tail amputation (P<0.05), outperforming PER977. Furthermore, compound 1 at 20 mg / kg achieved an inhibition rate of 72.1% against liver laceration-induced bleeding, and compound 1 at 20 mg / kg also significantly shortened WBCT (P<0.001). Compared to the control group, no significant changes were observed in bleeding volume or WBCT in the compound 1 control group (compound 1 20 mg / kg + NS). [Examples]

[0050] Antagonistic effect against heparin Experimental procedure: Tail amputation bleeding model: SD rats, excluding the control group and the 20 mg / kg + NS control group of compound 1, were administered heparin intravenously, followed immediately by intraperitoneal injection of 10% chloral hydrate. Anesthetized animals were placed on an electric blanket preheated to 37°C. Within 30 minutes, the jugular vein was isolated, a cannula was inserted, and saline and the drug were injected into the jugular vein. Five minutes after drug administration, an incision was made 5 mm from the tail end of the rat, and the bleeding loss (BL) over 15 minutes was recorded. Temperatures, including room temperature and water bath temperature, were strictly controlled during the experiment.

[0051] Liver laceration bleeding model: SD rats, excluding the control group and the 20 mg / kg + NS control group of compound 1, were administered heparin intravenously, followed immediately by intraperitoneal injection of 10% chloral hydrate. Anesthetized animals were placed on an electric blanket preheated to 37°C. Within 30 minutes, the jugular vein was isolated, a cannula was inserted, and 30 minutes after aspirin administration, the test substance was injected into the jugular vein. Five minutes after drug administration, the abdominal cavity was opened, and three incisions approximately 1 cm in diameter and 2 mm deep were made on the surface of the liver. Blood seeping from the liver was wiped away with pre-weighed dry cotton, and the bleeding loss (BL) over 30 minutes was recorded.

[0052] The experimental results are shown in Table 4 below. [Table 4]

[0053] The experimental results revealed the following: Compared to the control group, the solvent group showed a significant increase in induced bleeding in cases of tail amputation and liver laceration (P<0.05, P<0.001), with liver laceration bleeding increasing 5.5 times. Compared to the solvent group, compound 1 at 10 mg / kg significantly reduced the increase in induced rat bleeding in cases of tail amputation and liver laceration (P<0.05, P<0.01), achieving an inhibition rate of 88.9% against induced rat bleeding in liver laceration. Furthermore, when administered at the same dose, compound 1 showed superior inhibitory effect on rat tail amputation bleeding compared to PER977. Compared to the control group, no significant change in bleeding was observed in the compound 1 control group (compound 1 20 mg / kg + NS). [Examples]

[0054] Antagonistic effect against enoxaparin Experimental procedure: Tail amputation bleeding model: SD rats, excluding the control group and the compound 1 5.0 mg / kg + NS control group, were administered enoxaparin intravenously via tail vein, followed immediately by intraperitoneal injection of 10% chloral hydrate. Anesthetized animals were placed on an electric blanket preheated to 37°C. Within 30 minutes, the jugular vein was isolated, a cannula was inserted, and saline and the drug were injected into the jugular vein. Five minutes after drug administration, an incision was made 5 mm from the tail end of the rat, and the bleeding loss (BL) over 15 minutes was recorded. Temperatures, including room temperature and water bath temperature, were strictly controlled during the experiment.

[0055] Liver laceration bleeding model: SD rats, excluding the control group and the compound 1 5.0 mg / kg + NS control group, were administered enoxaparin intravenously via tail vein, followed immediately by intraperitoneal injection of 10% chloral hydrate. Anesthetized animals were placed on an electric blanket preheated to 37°C. Within 30 minutes, the jugular vein was isolated, a cannula was inserted, and the test substance was injected into the jugular vein 30 minutes after aspirin administration. Five minutes after drug administration, the abdominal cavity was opened, and three incisions approximately 1 cm in diameter and 2 mm deep were made on the surface of the liver. Blood seeping from the liver was wiped away with pre-weighed dry cotton, and the bleeding loss (BL) over 30 minutes was recorded.

[0056] The experimental results are shown in Tables 5-6 below. [Table 5]

[0057] [Table 6]

[0058] The experimental results revealed the following: Compared to the control group, the solvent group showed a significant increase in induced hemorrhage in cases of liver lacerations in animals (P<0.001), with liver laceration hemorrhage increasing sevenfold. Compared to the solvent group, compound 1 significantly reduced induced hemorrhage in rats after tail amputation, and the inhibitory effect of the 5 mg / kg group was superior to that of the PER977 (20 mg / kg) group. In the 1 mg / kg and 5 mg / kg dose groups of compound 1, induced hemorrhage in cases of liver lacerations in rats was significantly reduced (P<0.001), with inhibition rates reaching 62.7% and 89.1%, respectively. The inhibition rate of the 5 mg / kg group was equivalent to that of the PER977 20 mg / kg group. Compared to the control group, no significant change in hemorrhage was observed in the compound 1 control group (compound 1 5 mg / kg + NS).

[0059] The experimental results described above clearly show that the compound produced by the present invention has superior anticoagulant activity compared to PER977.

[0060] The above description of the embodiments is provided to facilitate understanding of the method and essence of the present invention. Those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications shall also be within the scope of protection of the claims.

Claims

【Request Item 1】 【Chemistry 1】 (wherein X, X', and X'' are independently selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups.) Y, Y', and Y'' are independently selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups. Z, Z', and Z'' are independently selected from the group consisting of molecular fragments containing heteroatoms that can be protonated under physiological conditions. A compound having the structure represented by the above formula I, or a salt thereof, or a deuterated compound thereof.

2. The anticoagulant reversal agent according to claim 1, characterized in that the alkyl group has 1 to 10 carbon atoms, the alkenyl group has 2 to 10 carbon atoms, and the heterocyclic group has 2 to 12 carbon atoms.

3. The Y-Z, Y'-Z', and Y''-Z'' are independently selected from the residues of a basic amino acid. Preferably, the compound according to claim 1, wherein the basic amino acid residue is a histidine residue, an arginine residue, or a lysine residue.

4. The compound according to claim 1, characterized in that the molecular fragment containing the heteroatom is selected from the group consisting of an amino group, a guanidine group, and an imidazole group. 【Request Item 5】 【Chemistry 2】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ and R ６ (These are independently selected from the group consisting of substituted or unsubstituted C1-C6 alkyl groups, C2-C6 alkenyl groups, and monocyclic or heterocyclic groups.) The compound according to claim 1, characterized by having a structure represented by the above formula I-a. 【Request Item 6】 【Chemistry 3】 The compound according to claim 1, characterized by having the above structure. 【Request Item 7】 【Chemistry 4】 The compound according to claim 1, characterized by having the above structure.

8. Step S1 involves reacting xanthine with a halide represented by the following formula a to obtain intermediate 1, Step S2 involves deprotecting the amino group of intermediate 1, Step S3 involves reacting the deprotected compound with the compound represented by the following formula b to obtain compound 2, The process includes step S4, which involves deprotecting compound 2 to obtain a compound represented by formula I. 【Transformation 5】 (wherein X is selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups) Y is selected from the group consisting of substituted or unsubstituted alkyl groups, alkenyl groups, and heterocyclic groups. Z is selected from the group consisting of molecular fragments containing heteroatoms that can be protonated under physiological conditions. A method for producing the compound according to any one of claims 1 to 7.

9. An anticoagulation reversal composition comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable adjuvant.

10. The use of a compound according to any one of claims 1 to 7 or an anticoagulant reversal composition according to claim 9 in the manufacture of a pharmaceutical product for preventing, treating or reducing bleeding caused by an anticoagulant, Preferably, the anticoagulant is one or more of a vitamin K antagonist and an indirect thrombin inhibitor. More preferably, the vitamin K antagonist is warfarin, and the indirect thrombin inhibitor is heparin or low molecular weight heparin.

11. A kit comprising a compound according to any one of claims 1 to 7 and one or more anticoagulants, Preferably, the anticoagulant is a vitamin K antagonist or an indirect thrombin inhibitor. A kit wherein the vitamin K antagonist is warfarin, and the indirect thrombin inhibitor is heparin or low molecular weight heparin.

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